Optimizing purification processes of recombinant outer membrane protein (OMP) of Candidatus Liberibacter asiaticus causing agent of citrus greening

Document Type : Research Paper

Authors

1 Department of Horticulture Science, Biotechnology & Plant Molecular Genetic Section, University of Hormozgan, Bandar Abbas, Iran

2 Corresponding author. Associate Professor, Department of Horticulture Science, Biotechnology & Plant Molecular Genetic Section, University of Hormozgan, Bandar Abbas, Iran

3 Department of Plant viruses, Iranian Research Institute of Plant Protection, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.

4 Associate Professor, Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

Abstract

Objective
The omp (Outer membrane protein) protein located at outer surface of the bacterial membrane body agent citrus greening disease (Candidatus Liberibacter asiaticus). The mentioned-protein can be used as antigen for generating polyclonal and monoclonal antibodies. Therefore, the purification of membrane proteins is exposed to many problems due to presence of hydrophilic structure. The purpose of this study was optimizing recovery and purification of the recombinant omp protein.
Materials and Methods
The pET28a bacterial expression construct containing omp gene was used to recombinant production of the mentioned-protein. The omp gene expression was conducted in E. coli strain Rosetta-Gami2. With considering to be hydrophilic of omp protein, its purification was investigated under denature condition. To attain it, the different concentrations of NaCL, urea and guanidine at the different acidity pH condition was applied. To obtain the pure omp recombinant protein, the protein recovery from polyacrylamide gel was performed by methods based on elution by diffusion, sonication, and electroelution. The evaluation of quality and quantity purified protein was analyzed by SDS-PAGE and the protein concentration was calculated by ImageJ software. Also, to validate the purified proteins, Western blot was applied by specific antibody.
Results
Primary results showed that using the 500 mM NaCL was detected as an essential share in purification buffers. Also, using urea as denature agent had twice the efficiency on the quantity of purified protein more than guanidine hydrocholoride and thus, when 8 m urea supplemented in purification buffer, the highest purified protein was attained (450 µl/ml). Furthermore, the results related to recovery the single band of protein from gel indicated that the electroelution has highest efficient on recovery of the target protein from gel matrix compared to sonication and elution by diffusion methods. The results of SDS-PAGE confirmed to appear a protein with 34 kDa related to molecular weight of target protein. Western blot validated the recombinant protein in membrane. 
Conclusions
The current study showed that using urea in 8 M, NaCL in 500 mM concentration in purification buffers following adjusting the elution buffer on pH (4) in elution purification step can be apply as an optimized condition to attain the highest target protein concentration. Furthermore, electroelution method introduced as efficient method in order to recovery band of the target protein from polyacrylamide gel matrix.

Keywords

References

 
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Agricultural Biotechnology Journal
Volume 14, Issue 1
April 2022
Pages 175-196

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